A new technique has become available for the rapid separation of two distinct populations of synaptosomes from brain. This project examined a variety of parameters to determine whether there might be functional differences between these 2 populations. The content of endogenous dopamine and serotonin differed between the fractions isolated from rat striatum. Fraction 6 contained more serotonin, while fraction 8 contained more dopamine than the corresponding fraction of synaptosomes. Similar extents of release of endogenous neurotransmitters was achieved on depolarization of the two fractions. Maximal rates of activity of the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, was not significantly different between the two populations. When the distribution of phosphoproteins in the two populations was examined, the majority of phosphoproteins showed no selective enrichment, while several were selectively localized in one or other population. Two proteins were identified: tyrosine hydroxylase was found selectively enriched in fraction 8, while DARPP-32 was enriched in fraction 6. The data suggests that a relative enrichment of dopaminergic and serotonergic synaptosomes into separate pools has been achieved.